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目的 :构建鼠源TRAIL(mTRAIL)真核表达质粒。研究其体内、外表达对肝癌细胞的诱导凋亡作用 ,抑制肝癌生长作用及与重组人FN多肽真核表达质粒pCH5 10协同抑制肝癌生长作用。方法 :采用RT PCR及重组DNA技术构建mTRAIL真核表达质粒 ;体内、外进行基因转染 ;用流式细胞仪检测肝癌细胞凋亡率 ,用TdT mediateddUTPnickendlabeling (TUNEL)法及免疫组织化学技术检测肝癌细胞凋亡 ;小鼠实体瘤块模型研究基因转染抑制肝癌生长作用。结果 :用RT PCR方法自小鼠脾细胞RNA扩增出TRAIL基因的全长cDNA ,并克隆至真核表达载体pcDNA3 .1中获重组质粒pX1;以pX1转染BHK细胞株后攻击肝癌细胞株H2 2细胞 ,可检测到肝癌细胞凋亡 ;肌肉内注射转染质粒DNApX1,通过诱导肿瘤细胞凋亡抑制肝癌生长 ;pX1与FN多肽真核表达质粒pCH5 10有协同抑制肝癌生长作用。结论 :质粒pX1可在细胞及小鼠体内表达 ;体内、外表达可诱导肝癌细胞凋亡并可通过该机制抑制肝癌生长 ;与FN多肽真核表达质粒pCH5 10有协同抑制肝癌生长作用
Objective : To construct a murine TRAIL (mTRAIL) eukaryotic expression plasmid. To study the in vitro and in vivo apoptosis-inducing effects of hepatoma cells, inhibit the growth of hepatocellular carcinoma, and synergize with the eukaryotic expression plasmid pCH5 10 of recombinant human FN polypeptide to inhibit the growth of hepatocellular carcinoma. Methods: RT-PCR and recombinant DNA technology were used to construct mTRAIL eukaryotic expression plasmid; gene transfection was performed in vitro and in vivo; flow cytometry was used to detect the apoptosis rate of hepatocellular carcinoma cells; TdT mediated dUTP Nickend labeling (TUNEL) method and immunohistochemical technique were used to detect liver cancer. Apoptosis; Mouse solid tumor block model study gene transfection inhibits the growth of liver cancer. Results: The full-length cDNA of TRAIL gene was amplified by RT PCR from mouse spleen cells and cloned into the eukaryotic expression vector pcDNA3.1 to obtain the recombinant plasmid pX1; the BHK cell line was transfected with pX1 and then challenged with the hepatoma cell line. In H2 2 cells, apoptosis of hepatocellular carcinoma cells can be detected. Intramuscular injection of plasmid DNA pX1 can inhibit the growth of hepatocellular carcinoma by inducing tumor cell apoptosis. pX1 and FN polypeptide eukaryotic expression plasmid pCH5 10 can synergistically inhibit the growth of hepatocellular carcinoma. Conclusion: Plasmid pX1 can be expressed in cells and mice; in vivo and in vitro expression can induce apoptosis of hepatoma cells and can inhibit the growth of hepatocellular carcinoma through this mechanism; and the eukaryotic expression plasmid pCH5 10 of FN polypeptide can synergistically inhibit the growth of hepatocellular carcinoma.